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Free Radical Biology & Medicine, Vol. 35, No. 8, pp.

910 921, 2003 Copyright 2003 Elsevier Inc. Printed in the USA. All rights reserved 0891-5849/03/$see front matter

doi:10.1016/S0891-5849(03)00436-2

Original Contribution
EPICATECHIN AND ITS METHYLATED METABOLITE ATTENUATE UVAINDUCED OXIDATIVE DAMAGE TO HUMAN SKIN FIBROBLASTS
SHARMILA BASU-MODAK,* MATTHEW J. GORDON,* LAURA H. DOBSON,* JEREMY P. E. SPENCER, CATHERINE RICE-EVANS, and REX M. TYRRELL*
*Department of Pharmacy and Pharmacology, University of Bath, Bath, UK; and Wolfson Centre for Age-Related Diseases, GKT School of Biomedical Sciences, Kings College, London, UK (Received 17 March 2003; Revised 19 June 2003; Accepted 27 June 2003)

AbstractThe ultraviolet A component of sunlight causes both acute and chronic damage to human skin. In this study the potential of epicatechin, an abundant dietary avanol, and 3'-O-methyl epicatechin, one of its major in vivo metabolites, to protect against UVA-induced damage was examined using cultured human skin broblasts as an in vitro model. The results obtained clearly show that both epicatechin and its metabolite protect these broblasts against UVA damage and cell death. The hydrogen-donating antioxidant properties of these compounds are probably not the mediators of this protective response. The protection is a consequence of induction of resistance to UVA mediated by the compounds and involves newly synthesized proteins. The study provides clear evidence that this dietary avanol has the potential to protect human skin against the deleterious effects of sunlight. 2003 Elsevier Inc. KeywordsUVA radiation, Flavanoids, Epicatechin, Cell death, Necrosis, Heme oxygenase-1, Free radicals

INTRODUCTION

Ultraviolet A (UVA) radiation (320 380 nm), a component of the solar UV spectrum, penetrates through the dermis and beyond to the subcutaneous tissue and affects both the epidermal and dermal components of skin [1]. The deleterious effects of UVA are manifested in human skin as erythema, photoaging, and skin cancer (reviewed in [2 4]). At the cellular level, UVA radiation causes signicant oxidative stress due to generation of reactive oxygen species such as singlet oxygen, hydroxyl radical, superoxide anion, and hydrogen peroxide, and the release of free iron, but most of the cellular effects of UVA irradiation seem to be mediated by singlet oxygen (reviewed in [57]). Constitutive cellular defenses against UVA-induced damage include simple antioxidant molecules (glutathione, carotenoids, ascorbate, and -tocopherol), and proteins (ferritin, heme oxygenase, glutathione peroxidase, superoxide dismutase, catalase). When cellular defenses are overwhelmed, UVA-induced oxidative damage becomes visible as depletion of intraAddress correspondence to: Professor R. M. Tyrrell, Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, UK; Tel: 44 (1225) 386793; Fax: 44 (1225) 383408; E-Mail: r.m.tyrrell@bath.ac.uk. 910

cellular glutathione [8], and oxidation of nucleic acids, proteins and membrane lipids (reviewed in [9]). Damaged cells respond by inducing a variety of genes in keratinocytes and broblasts that are implicated in both acute and chronic responses to this oxidative stress (reviewed in [10]). Extensive cellular damage results in cell death, which could occur either by apoptosis or necrosis in skin cells [7,11,12]. The current approach to protection against solar UVinduced oxidative damage to human skin relies heavily on either avoidance of excessive sunlight or the use of sunscreens, but dietary sources of antioxidants have the potential to complement these strategies. Plant polyphenols, especially the avonoids, constitute an important dietary source of antioxidants (reviewed in [13,14]). The catechin/avanol families of avonoids are major constituents of green tea, wines, apples, and chocolate. Recent studies that determined the avanol content in foods and beverages commonly consumed in the Netherlands showed that epicatechin (EC) was the abundant avanol in a wide variety of fruits, vegetables, and beverages [15,16]. After dietary intake, avanols can undergo conjugation and metabolism in the small intestine and liver involving glucuronidation, methylation, and sulphation ([17], reviewed in [18]). They also undergo colonic me-

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tabolism [19]. Increased plasma concentrations of EC as well as sulphate, glucuronide, and sulphoglucuronide conjugates of EC and its methylated metabolite have been observed in human volunteers after dietary intake [20 22]. These metabolites may contribute to the protective effects of dietary avonoids. Although it is not known yet whether EC and its metabolite 3'-O-methyl epicatechin (MeOEC) accumulate in human skin, radioactivity derived from orally administered [3H]( )epigallocatechin gallate has been detected in the skin of male and female mice [23], a nding which supports the possibility that EC and MeOEC could accumulate in human skin. Epigallocatechin gallate, the most abundant avanol in green tea, has been studied extensively in vivo in both rodents and humans, and has been found to have protective effects against UVB-induced inammatory responses, immune-suppression, intracellular generation of hydrogen peroxide, and skin carcinogenesis ([24,25], reviewed in [26,27]). Such studies have not employed EC. However, in in vitro studies, pretreatment with EC was found to protect cultured human skin broblasts, primary murine cortical neurons, and a hepatocyte cell line against oxidative damage induced by hydrogen peroxide [28 30], as well as primary striatal neurons against oxidative stress induced by oxidized LDL [31]. Interestingly, the in vivo metabolite, MeOEC, was shown to be equally protective against oxidative stress in broblasts and neurons, suggesting that the mechanism does not involve conventional antioxidant activity [29,31]. In contrast, the glucuronidated compounds elicited no cytoprotective effect. In the current study the ability of EC and one of its major in vivo metabolites, MeOEC, to protect against UVA-induced oxidative damage to cultured human skin broblasts was examined. Pretreatment with either of these compounds protects these cells against UVA-induced cell damage and cell death. This increased resistance to UVA-induced damage requires protein synthesis and seems to be largely independent of the antioxidant properties of EC.

Biochemicals (Lewes, UK). TRIZOL reagent was purchased from Invitrogen Ltd. (Paisley, UK). MTT, neutral red, propidium iodide, ( )- epicatechin and puromycin were purchased from Sigma (Poole, UK). Epicatechin was further puried by preparative HPLC. L-[4, 5-3H] Leucine (specic activity 120 190 Ci/mmol) was purchased from Amersham (Buckinghamshire, UK). 3'-Omethyl epicatechin was synthesized as described previously [29]. Cell culture Normal human skin broblasts FEK4 [32] were cultured routinely at 37C in 5% CO2 in Minimum Essential medium with Earles salts (EMEM) supplemented with 2 mM L-glutamine, 50 u/ml penicillin, 50 g/ml streptomycin, 0.2% sodium bicarbonate, and 15% FCS as described previously [33]. For all experiments, cells were used between passages 11 and 15. Cell treatments were carried out at 37C in a humidied CO2 incubator. Treatment of cells with avanols and UVA irradiation Stock solutions of the avanols were prepared in 50% methanol and stored at 80C until use. Aliquots were thawed only once for use in order to avoid degradation of the compound. FEK4 cells were seeded in 3 cm dishes (5 104 cells/dish) in complete EMEM and approximately 30 h later the medium was replaced with Fibroblast Growth medium (FGM) containing 5 g/ml insulin, 1 ng/ml basic broblast growth factor, 50 ng/ml amphotericin B, and 50 g/ml gentamicin. The cell monolayers were incubated in this medium (2.4 ml/dish) for 24 h before addition of either EC or MeOEC. For treatment, the volume of the conditioned medium was reduced to 1 ml and the excess medium was placed at 37C. The compounds were added to the required nal concentration such that the vehicle never exceeded 0.1% in the medium in order to avoid cellular effects of the vehicle itself. The highest concentration of EC and MeOEC that could be used was dictated by the solubility of the compounds in the vehicle. Control cells were pretreated cells with the vehicle alone. Cells were incubated with the compound for approximately 18 h and then irradiated with 500 kJm 2 UVA (unless specied) in Ca2 /Mg2 PBS (1 ml/dish containing 0.5 g/ml each of CaCl2 and MgCl2) using a broad spectrum UVA lamp (350 450 nm, Sellas, Germany) as described previously [34]. After irradiation the conditioned medium was added back (1 ml/dish) and the monolayers were incubated further for either 30 min (for MTT and Neutral red assays) or 18 h (for ow cytometry) and then analyzed. The compounds were not included during irradiation and postirradiation.

MATERIALS AND METHODS

Materials Routine tissue culture reagents were purchased from Gibco Invitrogen Ltd. (Paisley, UK). Fetal calf serum Gold was purchased from PAA Laboratories (Somerset, UK). Fibroblast growth medium and Minimum Essential Medium with Earls salts (without L-leucine and Lglutamine) were purchased from PromoCell (Heidelberg, Germany). LightCycler DNA Master SYBR Green I and Annexin-V-Fluos were purchased from Roche Molecular

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MTT assay The MTT assay [35,36] is a commonly used sensitive, quantitative, and reliable assay for measuring viability of cells. This assay is often considered as an indicator of mitochondrial function, but it has been established [37] that most of the MTT reducing activity is present in the endoplasmic reticulum that separates as the microsomal fraction after cell fractionation. After the postirradiation incubation of 30 min, cell monolayers were washed twice with warm PBS and 500 l of serum-free-EMEM containing 0.5 mg/ml MTT was added to each dish. Cells were incubated with the substrate for 2.5 h at 37C in a CO2 incubator. The substrate-containing medium was removed at the end of the incubation and 500 l of DMSO was added per dish to dissolve the formazan crystals. The dishes were agitated on a rocking platform for at least 10 min at ambient temperature, after which the absorbance of 100 l aliquots was measured against DMSO at 550 nm in a microplate reader (MR5000 Dynatech Laboratories, West Sussex, UK) and considered as the MTT reduction activity. The absorbance of the irradiated samples was expressed as a percent of the corresponding vehicle/compound-treated shams and plotted as percent activity in graphs. This activity was also used for estimation of fold increase in cytoprotection with compound treatment by assigning the activity in vehicle-treated samples a value of 1. Neutral red assay NR is a water soluble, weakly basic dye that passes across the plasma membrane passively and accumulates in the lysosomes of live cells [38,39]. In damaged cells the dye is no longer retained by the lysosomes and is lost from the cells, as the plasma membrane does not act as a barrier. As lysosomal membranes are also damaged in UVA-irradiated cells, the NR assay was chosen as an alternative assay for measuring damage in irradiated cells. After the 30 min postirradiation incubation, the conditioned medium in each dish was replaced with 500 l of neutral red medium (50 g/ml neutral red dye in fresh EMEM) and cells were placed for 1.5 h in a CO2 incubator at 37C. The neutral red medium was prepared on the day of the experiment, incubated at 37C for 30 min, and centrifuged at 3000 rpm for 10 min to clear any precipitate. This medium was then lter sterilized and added to the cells. After incubation, the neutral red medium was removed and cell monolayers were xed for 1 min with 500 l of xing solution (10% CaCl2, 0.4% formaldehyde). The dye was extracted from the cells by addition of 500 l of extraction solution (19% acetic acid, 50% ethanol) and 10 min agitation on a rocking platform at ambient temperature. The absorbance of 200

l aliquots was measured at 550 nm in a microplate reader (MR5000 Dynatech Laboratories). The absorbance in the irradiated samples was expressed as a percent of the corresponding vehicle/compound-treated shams and the value for percent retention was considered as an indicator of cell viability, as the dye is taken up actively into the lysosomes of live cells. The fold increase in cytoprotection was calculated as described for the MTT assay. Flow cytometry This technique allows quantication of live, apoptotic, and necrotic cells on a single-cell basis in cell populations. Distinction between necrotic and apoptotic cell death can be achieved reliably by combining a specic marker of apoptosis with a DNA stain. Fluoresceinconjugated Annexin V (AV) and the DNA stain propidium iodide (PI) were employed as markers of apoptosis and necrosis. AV, a Ca2 -dependent phospholipid binding protein with high afnity for phosphatidyl serine (PS), stains specically externalized PS in the plasma membrane of apoptotic cells (reviewed in [40,41]). The DNA stain PI enters cells only when the plasma membrane is damaged. FEK4 cells were incubated for 18 h after irradiation and the medium was collected to harvest detached cells. Cell monolayers were trypsinized with 0.25% trypsin/0.6 mM EDTA (0.5 ml/dish). After inactivation of trypsin with an equal volume of complete EMEM, the cell suspensions from two dishes were pooled with the corresponding reserved medium to constitute a sample. Cells were stained with Annexin-V-Fluos (AV) and propidium iodide (PI) as described in the manufacturers protocol (Roche Molecular Biochemicals) with slight modication as follows. Cell pellets were washed twice with incubation buffer, resuspended in 100 l of the same buffer, and 2 l of AV was added. Staining was carried out in the dark at ambient temperature for 15 min, after which 400 l of incubation buffer containing 5 g/ml propidium iodide was added. Flow cytometry was performed in a Becton Dickinson FACS Vantage (Cellquest version 3.3 software, Belgium) calibrated with Fluoresbrite beads and set up for electronic compensation of the emission spectra. Samples were analyzed using 488 nm excitation and detection in FL1 channel for AV (520 nm bandpass lter for uorescein) and FL3 channel ( 620 nm longpass lter) for PI. Live (AV /PI ), apoptotic (AV /PI ), and necrotic (total PI ) cell populations were determined in a total of 10,000 cells and expressed as percent. The apoptotic and necrotic cells in sham samples were subtracted from the corresponding populations in irradiated samples in order to exclude the low levels of cell death unrelated to UVA.

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RNA extraction Cell monolayers were lysed in the culture dishes (1 ml TRIZOL reagent/dish) at 4 h postirradiation and lysates from 3 dishes were pooled to constitute a sample. Glycogen was added to the lysates at a nal concentration of 50 g/ml in order to increase RNA yields. Total cellular RNA was extracted according to the manufacturers protocol (Invitrogen Life Technologies). Real time RT-PCR. A two-step RT-PCR approach was used to analyze HO-1 mRNA accumulation. cDNA synthesis. Total RNA (1 g/sample) was reverse transcribed using SuperScript rst-strand synthesis kit (Invitrogen Life Technologies) according to the manufacturers protocol. Random hexamers were used for priming the reaction to obtain a better representation of the RNA population in the cDNA samples. A 1:10 dilution of each cDNA was freshly prepared in PCR-grade water before each run and 2 l was used in each PCR reaction. Primers. The Heme oxygenase-1 (HO-1) primer pair [42] 5'-AAG AGG CCA AGA CTG CGT TC-3' (forward) and 5'-GGT GTC ATG GGT CAG CAG C-3' (reverse) gave an amplicon size of 74 bp in the human HO-1 cDNA sequence. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene and the primer pair [43] 5'-GAC ATC AAG AAG GTG GTG AA-3' (forward) and 5'-TGT CAT ACC AGG AAA TGA GC-3' (reverse) had an amplicon size of 178 bp in the human GAPDH cDNA. Desalted primers were obtained from Life Technologies, Paisley, UK and used for PCR reactions without further purication. Real time PCR. Real time PCR reactions were carried out in the LightCycler (Roche Molecular Biochemicals) using the uorescent dye SYBR Green I. The optimal MgCl2 concentration determined for both primer sets was 3 mM and each primer pair was used at a nal concentration of 0.5 M. The HO-1 and GAPDH mRNAs were analyzed in separate runs as amplication conditions optimized for the two primer sets were slightly different due to the amplicon sizes. PCR reactions were carried out in a volume of 20 l and the experimental protocol consisted of predenaturation, amplication, melting curve analysis, and cooling programs (LightCycler 3 Run version 5.32) with the following parameters for HO-1: the predenaturation step was a single cycle of 30 s at 95C. The amplication step consisted of 40 cycles of 0 s at 95C, 5 s at 55C, and 4 s at 72C, with uorescence measurement at the end of each cycle. The melting curve analysis comprised of 1 cycle of

0 s at 95C, 15 s at 65C, followed by a gradual increase to 95C (transition rate of 0.1C) with continuous uorescence acquisition. The default parameters of the cooling program were used, and unless specied, temperature transition rate of 20C/s was used in all the programs. The GAPDH experimental protocol was essentially the same except for the difference in the elongation time in the amplication cycle, which was 8 s at 72C. The standard curve for quantication of PCR samples was generated as follows. FEK4 cells were seeded in 10 cm dishes (6 105/dish) in complete EMEM and approximately 72 h later these were irradiated with 250 kJm 2 UVA. After irradiation the conditioned medium was added back to the culture dishes and these were incubated further for variable times up to 8 h in a 37C CO2 incubator. Real time PCR analysis of this time course of HO-1 mRNA accumulation showed that maximum levels were reached between 3 and 5 h postirradiation. Total RNA from 3, 4, and 5 h time point samples were reverse transcribed (4 g/sample) as mentioned above, and the cDNA samples were pooled to use as a standard. Serial dilutions in the range 1:10 to 1:3000 of this cDNA pool with arbitrary concentration values of 70 ng 0.23 g were used to generate separate standard curves for HO-1 and GAPDH. Each standard curve consisted of 6 dilution steps with a range of 3 orders of magnitude in the dilution series. Triplicates of each dilution were used for the standard curve runs and the data from these runs were used to create a coefcient le in the Relative Quantication software (Roche Molecular Biochemicals). A large batch of cDNA with a relatively high HO-1 signal was diluted 1:10 and used as calibrator for all experiments. Each sample run consisted of one set of experimental cDNAs and duplicates of the calibrator. The data le of each run was exported and analyzed in the Relative Quantication software using the dual mode with efciency correction. The normalized HO-1 mRNA values in the irradiated samples were expressed as fold increase of the corresponding controls.

Absorption spectra EC was diluted to a nal concentration of 30 M in Ca /Mg2 -PBS and irradiated at ambient temperature with 500 kJm 2 UVA in plastic disposable cuvettes with stoppers. The sham samples were left at room temperature for the time of irradiation. The absorption spectrum was measured immediately between 200 and 350 nm at 50 nm/min scan speed in a Kontron Instruments spectrophotometer (Uvikon 922, Milan, Italy). Samples were scanned in quartz cuvettes against Ca2 /Mg2 -PBS containing 0.06% methanol (vehicle).
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Protein synthesis FEK4 cells were seeded in 3 cm dishes and prepared for inhibitor treatment as described in an earlier section for EC treatment. Cells were treated in 1 ml with either 50 g/ml puromycin or with 0.06% methanol (vehicle) for 3 h at 37C, after which the medium was aspirated, cell monolayers were washed once with leucine-free medium (EMEM without L-leucine, but supplemented with L-glutamine, penicillin and streptomycin, and 0.5% FCS) and incubated in 1 ml of leucine-free medium for 1 h at 37C to deplete cells of leucine. After this depletion step, the medium was replaced with 500 l of labeling medium (20 Ci of 3H-Leucine/ml of leucinefree medium) and incubated further for 2 h at 37C. Either the vehicle or the antibiotic was included during the depletion and the labeling steps. Thus the total incubation time with the antibiotic was 6 h. After labeling, the medium was removed and cell monolayers were washed once with ice-cold PBS and lysed in 300 l of lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM EDTA, 1% Triton X-100, 1% SDS). Lysates (100 l/sample) were spotted on Whatman lter paper and air dried completely before TCA precipitation. Filters were incubated in 10% TCA for 10 min at 4C and then boiled in 5% TCA for 10 min. After this, they were incubated in 5% TCA at ambient temperature for 10 min, transferred to absolute ethanol at ambient temperature for 2 min and air dried at 37C for 1 h. TCA precipitable counts were measured in a liquid scintillation counter (Rack Beta 1209, LKB Wallace, Turku, Finland). The radioactivity incorporated into control samples was considered as 100% and that in antibiotic-treated samples was expressed as a percent of the controls. Statistical analysis Data were expressed as mean SD. Comparison of means of two groups of data was made using the unpaired, two-tailed Students t-test. Means of more than two groups of data in a graph were compared using one-way analysis of variance (ANOVA) with Tukeys honestly signicant difference (HSD) post hoc test in SPSS 10 for Windows software. Graphs of EC and MeOEC treatment were compared using univariate analysis of variance with Tukeys HSD post hoc test. Statistical signicance was determined at p .05.
RESULTS

ical prole indicating that the compounds are photostable (data not shown). Attenuation of UVA-induced cell damage EC and MeOEC were initially assessed for their effect on cellular damage in irradiated cells. Irradiation of FEK4 cells with 500 kJm 2 UVA decreased the MTT activity to approximately 40% that of control cells (Fig. 1A). Pretreatment with a range of concentrations (150 M) of either EC or MeOEC attenuated this loss in irradiated cells. The protective effect of EC was already observed at 1 M and reached a plateau at 10 M, whereas pretreatment with MeOEC resulted in a concentration-dependent increase in protection reaching steady state at 30 M. Although both compounds increased the cellular resistance to UVA, the effect of EC was more pronounced than that of MeOEC at 1 and 10 M. Overall comparison of the two curves using univariate ANOVA revealed that the increase in cellular resistance to UVA conferred by EC treatment was signicantly higher than by its metabolite MeOEC. Treatment with EC or MeOEC for 18 h had no effect on the MTT activity in sham irradiated cells, indicating that the compounds did not induce this enzyme activity (data not shown). The increased resistance to UVA observed in irradiated cells suggested cytoprotection by the compounds. The magnitude of cytoprotection is quantied as a fold increase over control MTT values in Fig. 1B (1.27 0.12 and 1.84 0.18 fold with 1 and 30 M EC, respectively). In order to conrm that this reected an increase in viable cells, the neutral red (NR) assay was also used and the fold increase in cytoprotection resulting from EC treatment was similar to that obtained with the MTT assay (Fig. 1B). Treatment with MeOEC did not result in cytoprotection at 1 M in either assay (Fig. 1C), whereas at 30 M a signicant increase in resistance by 1.6 0.08 and 1.91 0.41 fold was observed in the MTT and NR assays, respectively. The results in Fig. 1 show that both EC and MeOEC attenuate UVA-induced oxidative damage to cultured human skin broblasts. Cell death in UVA-irradiated broblasts UVA-induced apoptotic cell death has been observed in supercial dermal broblasts of human skin reconstructed in vitro [44]. Although it is generally observed that most cell death is necrotic in UVA-irradiated FEK4 cells [7], both types of cell death were monitored in this study using AV and PI staining. The cell population was visualized as a dot plot (Fig. 2A). Live cells are not stained for either PI or AV (lower left quadrant of Fig. 2A) because the plasma membrane is intact and PS is completely absent from the outer leaet. Apoptotic cells

Photostability of EC and MeOEC Control experiments on the effects of UVA were undertaken. In vitro irradiation of 30 M EC or MeOEC solutions in Ca2 /Mg2 -PBS with 500 kJm 2 UVA did not change the absorption spectrum or the HPLC analyt-

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Fig. 2. Kinetics of UVA-induced cell death. FEK4 cells were irradiated with UVA and analyzed for cell death by ow cytometry at various times postirradiation. (A) Dot plot showing typical categorization of live, apoptotic, and necrotic cell populations in irradiated samples. (B) The percent apoptotic and necrotic population was determined in 10,000 cells, corrected for background cell death and plotted against time. Data are means of 4 samples from independent experiments. The two types of cell populations at the various time points were compared to that obtained at 0 h using one-way ANOVA with Tukeys HSD post hoc test and statistical signicance (*) was determined at p .05. Fig. 1. Attenuation of UVA-induced oxidative damage. (A) FEK4 cells were treated with various concentrations of EC or MeOEC and irradiated with UVA. The MTT activity in irradiated samples was expressed as a percent of the corresponding sham and plotted against concentration of compound. Data are means of 6 samples from two independent experiments. EC and MeOEC graphs were compared using Univariate Analysis of Variance with Tukeys HSD post hoc test and statistical signicance (*) was determined at p .05. (B,C) The percent MTT activity obtained in control cells irradiated with UVA was considered as 1 and the activity in 1 and 30 M treated samples was expressed as a fold increase over controls. For the NR assay, the dye retention in irradiated samples was expressed as a percent of the corresponding sham. The fold increase in cytoprotection was calculated as for the

are AV but PI (lower right quadrant of Fig. 2A), as the asymmetry of the lipid bilayer of the plasma membrane is disturbed during the early stages of apoptosis and PS

MTT assay. Data are means of 5 samples from two independent experiments. The fold increase in cytoprotection in 1 and 30 M treated samples was compared to the controls (0 M) using one-way ANOVA with Tukeys HSD post hoc test and statistical signicance (*) was determined at p .05.

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Fig. 3. Modulation of UVA-induced cell death. FEK4 cells were treated with EC (A) or MeOEC (B), irradiated with UVA and analyzed by ow cytometry at 18 h postirradiation. The percentages of apoptotic and necrotic cell populations were corrected for background cell death. Data are means of 3 samples from two independent experiments. Each type of cell population at 1 and 30 M was compared to its corresponding control (0 M) using one-way ANOVA with Tukeys HSD post hoc test and statistical signicance (*) was determined at p .05.

accumulates in the outer leaet, whereas the plasma membrane remains intact. Necrotic cells are AV and PI , as in the late stages of apoptosis in vitro the plasma membrane becomes permeable to vital dyes. Usually this population is localized in the upper right quadrant of a dot plot. However, extensive membrane damage caused by UVA rapidly renders the plasma membrane permeable to vital dyes, resulting in a population of cells distributed in both the upper two quadrants (Fig. 2A). Therefore the total PI cells in the upper two quadrants were considered as necrotic. In sham-irradiated samples, the cell population consisted of approximately 90% live, 8% necrotic, and 2% apoptotic cells (data not shown), indicating that incubation in PBS and the staining procedure did not cause extensive cell death. The background of necrotic or apoptotic cell death was subtracted from the corresponding irradiated samples in all experiments. Because the value subtracted amounts to approximately 10%, the total of cells scored in irradiated samples never exceeds 90%. Cell death was quantied in FEK4 cells at different time points after irradiation with 500 kJm 2 UVA. Immediately after irradiation, 26 2% of the cells are identied as necrotic (Fig. 2B). This population increased signicantly to a maximum of 49 4% at 6 h post irradiation followed by a small decline to 40% at 24 h. The 5% of the population identied as apoptotic did not change signicantly up to 24 h after irradiation. Thus, cell death induced in FEK4 cells by UVA irradiation was mainly necrotic. Because the level of cell death was more or less constant between 6 and 24 h, the 18 h

time point was chosen to study the effect of EC and MeOEC on UVA-induced cell death. Modulation of UVA-induced cell death by EC and MeOEC The effect of pretreatment with 1 and 30 M EC or MeOEC on levels of live, necrotic, and apoptotic cells was examined in irradiated samples. Irradiation with 500 kJm 2 UVA resulted in death of approximately twothirds of the cell population (Fig. 3A and B). Treatment with 1 and 30 M EC increased the live cell population from 28 3% to 41 3 and 71 0.5%, respectively (Fig. 3A). A corresponding decrease from 63 3% to 47 4 and 19 4% was observed in the necrotic cell population. These changes were signicant for both the live and the necrotic cell populations. No effect of MeOEC was observed at 1 M (Fig. 3B), but 30 M led to signicant protection against cell death of an order similar to that observed for EC. The low level of the apoptotic cell population was unaffected in both cases. Thus pretreatment with the higher concentration of EC or MeOEC increased the cellular resistance to UVA by 2.52.8-fold. Kinetics of EC-mediated cytoprotection The correlation between time of treatment and appearance of resistance to UVA was investigated. FEK4 cells were treated with 30 M EC for 1, 3, and 6 h, irradiated with 500 kJm 2 of UVA radiation and then analyzed for cell death at 18 h postirradiation. The live cell population

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consistent with the idea that EC was inducing a protective response in irradiated cells. Requirement of protein synthesis for the EC response The question as to whether this protective response was dependent on de novo protein synthesis was investigated. Cells were pretreated with 30 M EC for 6 h, followed by irradiation with 500 kJm 2 of UVA and then ow cytometric analysis was undertaken at 18 h postirradiation. Puromycin, an inhibitor of protein synthesis, was tested on FEK4 cells. Incubation with a range of concentrations of puromycin (0 50 g/ml) for 6 h before irradiation had no signicant effect on the total dead cell population in irradiated samples (data not shown). The highest concentration of puromycin (50 g/ml) decreased protein synthesis to 6 7% in unirradiated cells as revealed by 3H-leucine incorporation in total proteins (data not shown). Incubation of cell monolayers with this concentration of puromycin for 6 h had no effect on either live or total dead cell populations in irradiated samples (Fig. 5A). Pretreatment with 30 M EC increased the resistance to UVA by 1.7-fold and an absence of protein synthesis during the EC-treatment had no effect on this increase. Protein synthesis was measured in cells 18 h after removal of the inhibitor, which corresponded to the time point of ow cytometric analysis. 3H-leucine incorporation was 49 6% (data not shown), indicating a partial reversal of protein synthesis inhibition. Therefore, in a further set of experiments, cell monolayers were treated with EC for 6 h and puromycin was added only after UVA irradiation. Presence of the

Fig. 4. Kinetics of the EC effect. Cells were treated with 30 M EC for various times, irradiated with UVA and analyzed for cell death at 18 h postirradiation by ow cytometry. Control cells were treated with the appropriate concentration of vehicle. Data are means of 3 samples from three independent experiments. The level of the live cell population at the various time points was compared to that at 0 h using one-way ANOVA with Tukeys HSD post hoc test and statistical signicance (*) was determined at p .05.

increased from 15 5% to 26 11%, 37 5% (2.5-fold) and 48 9% (3.3-fold) with 1, 3, and 6 h pretreatments, respectively (Fig. 4). This was accompanied by a corresponding decrease in the necrotic population (data not shown). The live and necrotic cell populations remained unchanged with variable time of vehicle treatment in control samples. These results are

Fig. 5. Effect of inhibition of protein synthesis on EC-mediated cytoprotection. (A) Cells were treated with 30 M EC and 50 g/ml puromycin for 6 h, irradiated with UVA and then incubated for a further 18 h without compound or inhibitor. Flow cytometry was performed and data plotted as means of 6 samples from three independent experiments. (B) Cells were treated with 30 M EC for 6 h and irradiated with UVA. After irradiation 50 g/ml puromycin was added back to the appropriate samples for the 18 h postirradiation incubation. Samples were analyzed by ow cytometry and data expressed as means of 4 samples from two independent experiments. Each type of treatment in the two cell populations was compared to its corresponding control using one-way ANOVA with Tukeys HSD post hoc test and statistical signicance (*) was determined at p .05.

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Fig. 6. Modulation of UVA-induced HO-1 mRNA accumulation by EC. FEK4 cells were treated with 30 M EC and irradiated with 50, 100, 250, and 500 kJm 2 UVA. Total cellular RNA was isolated at 4 h postirradiation and HO-1 mRNA was quantied using two-step RTPCR. GAPDH mRNA levels were determined in all samples and used for normalizing HO-1 mRNA levels. Data were expressed as a fold increase over corresponding shams and means of 3 samples from three independent experiments were plotted against UVA dose. The means of the two groups at 500 kJm 2 was compared using the unpaired, two-tailed Students t-test and statistical signicance (*) was determined at p .05.

protein synthesis inhibitor during the postirradiation incubation before ow cytometry abolished the protective effect of EC (Fig. 5B). Taken together, these results indicate that the cytoprotective effect of EC was dependent on protein synthesis. UVA-induced heme oxygenase-1 mRNA accumulation in EC-treated broblasts Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme catabolism, is a well-known example of an oxidant-inducible gene [45]. UVA radiation activates this gene via singlet oxygen generation [46] and cellular levels of HO-1 mRNA are used widely as a measure of cellular oxidative stress status. Pretreatment of cells with EC for 18 h before irradiation had no effect on the UVA-induction of HO-1 mRNA up to 250 kJm 2 (Fig. 6), indicating that singlet oxygen scavenging was an unlikely mechanism of protection. At higher doses there is a decline in HO-1 mRNA levels due to suppression of transcription [46]. EC treatment attenuated this decline signicantly (Fig. 6), presumably because it prevented the suppression of transcription by high doses of UVA.
DISCUSSION

These ndings report protective effects of EC and one of its in vivo metabolites, MeOEC, against UVA-in-

duced oxidative damage. The results clearly demonstrate that pretreatment of cultured human skin broblasts with either compound induces resistance to UVA-induced cell damage and cell death. The time dependence for development of the protective response and the requirement for protein synthesis, together with the observation of a similar protection using both EC and its methylated metabolite, support the notion that this is an adaptive response largely independent of the hydrogen-donating antioxidant properties of EC. The ow cytometry measurements undertaken in this study conrm that UVA radiation causes primarily necrotic cell death in human skin broblasts and that apoptosis is only a minor pathway of cell death. It has been proposed that damage generated by singlet oxygen in UVA-irradiated cells causes them to undergo apoptosis rapidly so that they are already in the secondary necrosis stage during irradiation [47]. Oxidative damage results in depletion of glutathione and ATP as well as extensive peroxidation of lipids, and these changes would promote onset of mitochondrial permeability transition, a common event in both types of cell death [48]. However, such depletion favors mitochondrial permeability transition towards necrotic rather than apoptotic cell death (reviewed in [49,50]). Furthermore an increased prooxidant state of cells would favor inactivation of caspases (reviewed in [50]). These factors are all consistent with the predominantly necrotic cell death actually observed in UVA-irradiated broblasts. Both EC and its methylated metabolite clearly protect against cell damage induced by UVA radiation as judged by simple cell damage assays (MTT and NR) in cultured human skin broblasts. This is broadly in agreement with results reporting hydrogen peroxide-induced oxidative stress in the same cell type [29] as well as murine cortical neurons [28]. Both compounds also protect against UVA-induced necrotic cell death in skin broblasts, whereas in the same cell type and in primary striatal neurons, they have been shown to protect against apoptotic cell death induced by hydrogen peroxide [28,29]. This would indicate that the mechanism of protection involves an early step common to both cell death pathways, such as prevention of initial damage. The protection against UVA-induced cell death could relate to the antioxidant properties of the compounds. However, this is inconsistent with the similar effects seen with both EC and its methylated derivative, because the latter shows much lower antioxidant activity [29]. Furthermore, the time dependence of the development of EC-mediated protection is indicative of an induction mechanism that eventually leads to the manifestation of an adaptive response. Consistent with this, de novo protein synthesis is required for the development of the protection. Interestingly, UVA irradiation of skin bro-

Epicatechin protects against UVA damage

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blasts in culture leads to an adaptive protection against subsequent oxidative membrane damage and this response is mediated through HO-1 [51]. High levels of HO-1 gene expression have since been observed in several pathological and inammatory conditions (reviewed in [52]) and in vitro and in vivo studies clearly show that this enzyme mediates a protective response against cell and tissue injury [53]. The signicantly higher levels of HO-1 mRNA observed at a UVA dose of 500 kJm 2 in EC-treated cells compared to the corresponding control are consistent with the possibility that this gene may be involved in the cytoprotection, but to examine this would require further testing in a HO-1 decient model. The gene targeted HO-1 mouse model [54] could prove useful for such studies. In addition to the similarity of the protective effects of EC and MeOEC, two other sets of experiments indicate that protection is not due to antioxidant properties of the compound. Firstly, cellular uptake of EC and MeOEC occurs within 2 h of treatment in FEK4 cells and the uptake does not change with 18 h treatment [28]. A direct antioxidant effect would be expected to result in a protective response that remained unchanged between 2 and 18 h EC treatment, and this was not the case in these experiments. Secondly, the dose-dependent accumulation of HO-1 mRNA, a widely used marker of oxidative stress [34], was used to test the involvement of an antioxidant mechanism. Because the dose-dependent accumulation of HO-1 mRNA was not suppressed in EC pretreated cells up to an intermediate dose of 250 kJm 2, this compound seems not to be acting by the prevention of the generation of active oxygen species or their interaction with critical targets. However, it should be noted that in an earlier study from this laboratory, epigallocatechin, another green tea avanol, was shown to decrease signicantly the UVA induction of HO-1 at 250 and 400 kJm 2 in FEK4 cells [55]. Certain structurally related differences between the compounds, such as the superior ability of epigallocatechin to chelate iron (CRE, unpublished studies), could inuence the response of this early marker of oxidative stress to UVA. Conclusions This study reveals a protective role of EC, an abundant dietary avanol, and one of its major in vivo metabolites (MeOEC) against UVA-induced damage and cell death in cultured human skin broblasts. The protection involves an adaptive response dependent on protein synthesis and is not mediated by photo-degradation products. Given the potential of this avanol as an agent for enhancing the protection of human skin against acute UVA damage, it would be of value to probe further into the mechanism of EC-induced resistance.

Acknowledgements This research was supported by a European Fifth Framework RTD programme Grant (Grant no. QLK4-199901590) in which C.R.-E. and R.M.T. are contracting partners.

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[42]

[23]

[43]

[24]

[44]

[25]

[45] [46]

[26]

[47] [48]

[27] [28]

[29]

[49]

[30]

[50]

[31]

[51]

[32]

[52]

[53]

[33]

[54]

[34]

[55]

[35]

[36]

[37]

ABBREVIATIONS

[38]

[39]

[40] [41]

ANOVAAnalysis of variance AVAnnexin-V-Fluos DMSODimethyl sulfoxide ECEpicatechin EDTAEthylenediaminetetracetic acid EMEMMinimum essential medium with Earles salts FCSFetal calf serum FGMFibroblast growth medium GAPDHGlyceraldehyde phosphate dehydrogenase HO-1Heme oxygenase-1 HSDHonestly signicant difference

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LDLLow-density lipoprotein MeOEC3'-O-methyl epicatechin MTT3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide] NRNeutral red PBSPhosphate-buffered saline

PIPropidium iodide PSPhosphatidyl serine RT-PCRReverse transcriptase-polymerase chain reaction TCATrichloroacetic acid UVAUltraviolet A (320 380 nm) radiation